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Abstract

Coiled-coil domain containing 134 (CCDC134), a characterized secreted protein, may serve as an immune cytokine and illustrates its potent antitumor effects by augmenting CD8+ T-cell-mediated immunity. Additionally, CCDC134 may also act as a novel regulator of human alteration/deficiency in activation 2a, and be involved in the p300-CBP-associated factor complex and affect its acetyltransferase activity. To clarify the biological and pathological function of CCDC134, the present study generated a viable and fertile Ccdc134fl/fl mouse strain that allowed temporal and spatial control of gene ablation. Ccdc134-/- embryos generated by crossing of Ccdc134fl/fl mice with human β-actin-Cre or zona pellucida 3-Cre transgenic mice were embryonic lethal from embryonic day (E)12.5 to birth. Ccdc134 loss was associated with severe hemorrhages in the brain ventricular space and neural tube, pale and abnormal livers, cardiac hypertrophy and placental distress. Furthermore, it was demonstrated that a fraction of E13.5 fetal livers and brains exhibited reduced cell proliferation and vascular endothelial cell defects. CCDC134 also exhibited a dynamic and specific expression pattern during embryo development. The present results suggest that Ccdc134 may have specific biological functions in regulating mouse embryonic development.

Introduction

Coiled-coil domain containing 134 (CCDC134) was
first identified through high-throughput functional screening
systems using an Elk1 trans-reporting system in the Peking
University Center for Human Disease Genomics (Beijing, China). Our
earlier study demonstrated that CCDC134 is a classical secreted
protein that inhibited Elk1 transcriptional regulation and
mitogen-activated protein kinase (MAPK) signal transduction through
the Raf-1/MEK/extracellular signal-regulated kinase and c-Jun
N-terminal kinase/stress-activated protein kinase pathways
(1). A previous study also
identified a role for CCDC134 in tumor development; CCDC134 was
identified as a candidate biomarker of malignant transformation
with decreased expression in gastric cancer, and targeted small
interfering RNA knockdown of CCDC134 promoted tumor migration and
invasion via the MAPK pathway (2).

CCDC134 was proposed to have immune cytokine
function, and directly promoted CD8+ T-cell activation,
proliferation and cytotoxicity (3). Additionally, CCDC134 demonstrated
its potent antitumor effects by augmenting CD8+
T-cell-mediated immunity (3).
Mechanistically, exposure to CCDC134 promoted CD8+
T-cell proliferation through the Janus kinase 3-signal transducer
and activator of transcription 5 pathway, and two members of the
γc cytokine family could effectively block CCDC134
binding to activated CD8+ T-cells, which provided
evidence that CCDC134 may serve as a potential member of the
γc cytokine family (3).

On the basis of CCDC134 molecular structure and
transcription regulatory capacity, it is suggested that CCDC134 is
also a nuclear protein that acts as a critical regulator of human
alteration/deficiency in activation 2a (hADA2a) to enhance the
stability of hADA2a and inhibit its proteasome-dependent
degradation (4). Additionally,
CCDC134 participated in the p300-CBP-associated factor (PCAF)
complex via hADA2a to affect its histone acetyltransferase (HAT)
activity, which primarily acetylated lysine 14 of H3, but also less
efficiently acetylated lysine 8 of H4 (5). Also, CCDC134 was involved in the
repair of ultraviolet-induced DNA damage (4). The above evidence indicates that
CCDC134 may function as a cytokine that mediates immune responses
and a nuclear protein, similar to high mobility group box
chromosomal protein 1, interleukin-1α (IL-1α) and IL-33 (6,7).

Thus, CCDC134 may serve as a multi-faceted
adaptor/scaffolding protein to relay cellular signals to the
cytoplasm and the nucleus. To determine if any of these cellular
mechanisms for CCDC134 may be biologically relevant and significant
in vivo, the present study generated a murine model carrying
a conditional allele for Ccdc134. The present study reports
the generation and first characterization of a germline
Ccdc134 null mutant allele in mice, which, when homozygous,
is embryonic lethal and may impair embryonic angiogenesis.

Materials and methods

Targeting the Ccdc134 allele

The Ccdc134 targeting vector was generated
with two flippase recognition target (FRT) sites flanked by a
neomycin (neo)-resistant cassette upstream of the existing 3′ loxP
site, and the 5′ loxP site was inserted into upstream exon 3, by
Nanjing Biomedical Research Institute of Nanjing University
(Nanjing, China), according to well-described principles and
methods (8). The targeting region
of the recombination vector and its relation with the
Ccdc134 locus are demonstrated in Fig. 1A. The targeting vector was
linearized with NotI (New England BioLabs, Inc., Ipswich,
MA, USA) and electroporated into B6/BLU embryonic stem (ES) cells.
G418-resistant clones were screened for homologous recombination by
long range polymerase chain reaction (PCR). Out of 64 clones, 4
were further identified with correct targeting by Southern blot
analysis.

The mice were housed and bred under pathogen-free
conditions at the Laboratory Animal Research Facility of Peking
University Health Science Center (Beijing, China). A total of 5
mice/cage were maintained under laboratory conditions at 25°C,
under a normal 12-h light/dark cycle with a humidity of 55% and
access to food and water ad libitum. Experimental procedures
were approved by the Institutional Animal Care and Use Committee of
Peking University Health Science Center (Beijing, China), following
the guidelines of the Care and Use of Laboratory Animals.

Genotypic analyses

DNA was extracted from ES cells using a genomic DNA
extraction kit (Qiagen China Co., Ltd., Shanghai, China) following
the manufacturer's protocol. Screening of ES cells was performed by
long range PCR analysis for 3′-end screening using a targeting
vector-specific forward primer (5′-GCATCGCATTGTCTGAGTAGGTG-3′) and
a reverse primer (5′-TCTTGCAGAGCAAGAGCGAG-3′) inside the targeted
region. In addition, a second pair of primers specific for 5′-end
screening outside of the target region was used forward,
5′-AACCTCACCCACTCTCTCACCG-3′ and reverse,
5′-AAGGGTTATTGAATATGATCGGA-3′. PCR analysis was performed using a
Takara LA Taq long PCR system (Takara Biomedical Technology Co.,
Ltd., Beijing, China) using conditions as follows: Denaturation at
94°C for 5 min; followed by 30 cycles of amplification at 94°C for
1 min, 55°C for 1 min and 65°C for 5 min; and a final extension
step at 72°C for 10 min.

High molecular weight genomic DNA was extracted from
ES cells, digested with either SpeI or SacI (New
England BioLabs, Inc.) and subjected to electrophoresis in 0.9%
agarose gel. For Southern blot analysis, genomic DNA was
transferred to a nylon membrane overnight, and then hybridized
overnight at 42°C using a 5′ external probe with
SpeI-digested DNA or 3′ external probe with
SacI-digested DNA specific to the Ccdc134 sequence
with 32P (GE Healthcare, Chicago, IL, USA) by a random
prime labeling method. Finally, the blot was monitored with
radioautograph to confirm homologous recombination.

Genomic DNA from tails of 3-wcditions as follows:
Denaturation at 94°C for 5 min; 35 cycles of amplification at 94°C
for 30 sec, 55°C for 30 sec and 72°C for 30 sec; and a final
extension step at 72°C for 5 min. Two types of Ccdc134 PCR
primers used for PCR analysis were as follows: p1,
5′-TCCTAACCCTGTCGCTCCCT-3′; p2, 5′-CCAGACAGAGGTGAGCTGCT-3′; p3,
5′-GCACCCTGAGCCAAGTTTAG-3′; and p4, 5′-CCTAACCTATGCCTCCAAAG-3′.
Genomic DNA from the targeted allele yields a 615-bp fragment with
a primer pair p1/p3, and a wild-type allele yields a 420-bp
fragment with primer pair p2/p3, or a 490-bp fragment with
p1/p4.

Total RNA was extracted from
Ccdc134+/+, Ccdc134+/− and
Ccdc134−/− embryos at different stages with
TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.,
Waltham, MA, USA), following the manufacturer's protocol. cDNA was
synthesized using a Revert Aid First Strand cDNA synthesis kit
(Fermentas; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA),
following the manufacturer's recommendations. The following PCR
cycling conditions were used: Denaturation at 94°C for 3 min; 35
cycles of amplification at 94°C for 30 sec, 55°C for 30 sec and
72°C for 30 sec; and a final extension step at 72°C for 5 min.
Ccdc134 primer sequences used for RT were as follows F1,
5′-GTTGGCACTGAAGAACCTGG-3′ and R1, 5′-ACGGGTTCCGGAAGTCAGAA-3′. The
qPCR analysis using SYBR-Green master mix was performed using an
ABI 7500 Real-Time PCR system (both from Applied Biosystems; Thermo
Fisher Scientific, Inc., Waltham, MA, USA) according to the
manufacturer's protocol (9). The
following primer sequences were used: Ccdc134 forward,
5′-GCTCCCTTCTCCCTGCAC-3′ and reverse, 5′-AGGCCACAGGAGGACAGA-3′; and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward,
5′-AAGAGGGATGCTGCCCTTAC-3′ and reverse, 5′-CCATTTTGTCTACGGGACGA-3′.
The mRNA expression levels of Ccdc134 were normalized to
GAPDH. All samples were assayed in duplicate, and average
values were used for quantification (9).

Histology and immunohistochemistry
analysis

Whole embryos at 13.5 days post coitus (E13.5) were
fixed in 10% formaldehyde solution at room temperature for over 24
h, embedded in paraffin, sectioned at 5-μm thickness,
stained with hematoxylin for 4 min, and stained with eosin for 2
min. All procedures were performed at room temperature for
histologic examination. Images were captured on a BX-53 inverted
fluorescence microscope (Olympus Corp., Tokyo, Japan) at different
magnifications (×2, ×10 and ×20), and processed with Adobe
Photoshop CS 5.0 (Adobe, San Jose, CA, USA).

Statistical analysis

All data were expressed as the mean ± standard
deviation. The differences among groups were analyzed using one-way
analysis of variance followed by Bonferroni correction. Statistical
analyses were performed using SPSS 11.0 (SPSS, Inc., Chicago, IL,
USA). P<0.05 was considered to indicate a statistically
significant difference.

Results

Generation of floxed Ccdc134 allele and
mice

To investigate further the biological role of
Ccdc134 in vivo, the present study attempted to generate
conditional Ccdc134-null mice using a Cre/loxP strategy. A
homologous targeting construct was prepared with the two loxP sites
flanking Ccdc134 exon 3–6, as well as a neo resistance
cassette (a positive selection marker) within intron 6, flanked by
FRT sites (Fig. 1A). Upon
transfection of ES cells with the linearized targeting vector and
G418 selection, 64 independent drug-resistant clones were selected
and screened for homologous recombination by long range PCR
analyses. The 5.4-kb fragment was amplified from the targeted
allelic genomic DNA with a primer pair for 3′-end screening,
whereas the 6.4-kb fragment was amplified with a primer pair for
5′-end screening (Fig. 1B). A
total of 17 clones were identified as potentially homologous
targeted lines. Additionally, 4 correctly targeted clones were
identified by Southern blot analysis (Fig. 1C).

To generate chimeras, two ES cell lines were
injected into blastocysts of C57BL/6J mice. Male offspring with a
high degree of chimerism were crossed with C57BL/6J females to
generate floxed Ccdc134 mice
(Ccdc134fln/wt). The neo locus was then removed
by crossing with FLP-expressing transgenic mice (10,11). Ccdc134 conditional allele
mice (Ccdc134fl/fl) were then generated by
intercrossing Ccdc134fl/wt mice, and their
genotyping was performed by PCR analyses using tail-derived DNA
(Fig. 1D). The transmission of
Ccdc134wt/wt, Ccdc134fl/wt and
Ccdc134fl/fl followed a Mendelian ratio, and
homozygous flox mice exhibited wild-type characteristics with
normal CCDC134 expression, reproductive capability and lifespan
(data not shown), suggesting that the flox alleles do not influence
Ccdc134 gene activity.

Ccdc134 deficiency is embryonically
lethal

The Ccdc134fl/fl mice were crossed
with ACTB-Cre transgenics, which expressed Cre recombinase under
the control of the ACTB gene promoter in all cells of the embryo by
the blastocyst stage of development, to generate Ccdc134
constitutive KOs, referred to as
ACTB-Cre-Ccdc134−/− (Fig. 1A) (12). Genotyping of progeny from
intercrossed Ccdc134 hetero-zygote
(ACTB-Cre-Ccdc134+/−) demonstrated that, among
284 pups born, 185 (65%) were heterozygous for a null allele and 99
(35%) were wild-type (Ccdc134+/+). No homozygous
mice were born, while heterozygous mice were present at the
expected Mendelian ratio (Table
I). Subsequently, another Cre line, the Zp3-Cre transgenic
line, was used in an attempt to delete Ccdc134 at an earlier
stage in embryonic development. Unlike ACTB, Zp3 is expressed in
the growing oocyte prior to the completion of the first meiotic
division (13). The
Ccdc134 heterozygotes (Zp3-Cre-Ccdc134+/−)
were generated by crossing female
Zp3-Cre-Ccdc134fl/wt mice with male wild-type.
Crosses between these heterozygous mice also delivered only
heterozygous (128) or wild-type (64) live pups in the ratio of 2:1,
consistent with prenatal lethality of
ACTB-Cre-Ccdc134−/− embryos (Table I). A representative genotype is
demonstrated in Fig. 2A. The
complete absence of Ccdc134 homozygous KO mice is a
statistically significant deviation from the expected ratio
(P<0.0001; data not shown), suggesting that a homozygous
Ccdc134-null genotype is embryonically lethal. Timed mating
indicated that mortality of Ccdc134−/− embryos
began between E11.5 and E12.5. The rate of mortality of
Ccdc134−/− embryos increased after E11.5 (0%
until E11.5, 16.67% at E12.5, 42.86% at E13.5 and 100% at E14.5)
and mortality was observed in all Ccdc134−/− mice
at E14.5 (Table I). These data
indicate that Ccdc134 deficiency causes embryonic lethality,
supporting a crucial role for Ccdc134 during
embryogenesis.

To confirm the inability of the targeted
Ccdc134 allele to support CCDC134 expression, the wild-type,
heterozygous and KO mice embryos at E13.5 were prepared and
analyzed by RT-PCR and western blot analysis. Ccdc134 mRNA
were not detected in Ccdc134−/− mice (Fig. 2B), and no CCDC134 protein was
detected with the use of a specific antibody to CCDC134 (Fig. 2C).

However, Zp3-Cre-Ccdc134+/− mice
appeared to be grossly normal. These heterozygotes bred without
difficulty and delivered normal sized pups. No histologic deficits
were observed. No apparent anatomic or microscopic features could
reliably discriminate heterozygotes from their wild-type
littermates. Previous study revealed that CCDC134 illustrated
potent antitumor effects by augmenting CD8+
T-cell-mediated immunity (3),
therefore different immune cell populations were analyzed in the
spleen and thymus. The results indicated no obvious difference
between heterozygotes and their wild-type littermates.
Additionally, B16 graft tumors were established in
Ccdc134+/− and Ccdc134+/+
controls, and it was demonstrated that Ccdc134+/−
mice may slightly accelerate tumor growth compared with wild-type
controls; however, no significant difference was observed (data not
shown). These data prompted us to restrict our efforts to the
comparison of the wild-type and Ccdc134−/−
populations, to define rigorously the mutant phenotype without
consideration of subtle or dose-dependent deficits.

Dynamic expression analyses of Ccdc134 in
whole embryos during embryogenesis

To determine whether Ccdc134 expression was
altered at various developmental stages of the embryo, the mRNA
expression level of Ccdc134 at four developmental stages was
compared. The expression level of Ccdc134 decreased from
E6.5 to E9.5, followed by an increasing trend from E9.5 to E12.5,
and then a decrease at E14.5. Thus, Ccdc134 mRNA showed the
highest expression level at E12.5 (Fig. 3A).

To gain further insight into the spatial and
temporal expression patterns of CCDC134 during embryonic
development, whole mount embryo sections were analyzed by
immunohistochemistry using specific rabbit anti-CCDC134 antibody.
This revealed histological details of CCDC134 expression in the
developing mouse embryo. At E6.5, the most prominent expression was
observed in the Reichert's membrane and endoderm. Furthermore,
CCDC134 expression was detected in the neural tube and
trophoblastic giant cells at E9.5. With the development of the
mouse embryo, prominent expression of CCDC134 was detected in the
somite and major organs, including the liver and lung, from E12.5
to E14.5 (Fig. 3B). At these
stages, CCDC134 was highly expressed in the somite and liver,
suggesting that CCDC134 participated in the development and
formation of major organs.

Morphological and histological pathology
of Ccdc134−/− embryos

To assess the role of CCDC134 throughout
development, Ccdc134+/+,
Ccdc134+/− and Ccdc134−/−
embryos were euthanized at E10.5–E16.5. Necropsies through this
interval demonstrated that Ccdc134−/− embryos
appeared similar to Ccdc134+/+ and
Ccdc134+/− littermates at E10.5, while
Ccdc134−/− embryos exhibited severe hemorrhaging
(100% penetrance) in the brain ventricular space and neural tube at
E13.5. Additionally, Ccdc134−/− embryos later
than E13.5 looked anemic, and their size was slightly smaller than
those of wild-type embryos (Fig.
4A). Subsequently, embryos at E16.5 were isolated, and the
absorbed embryos with genotypes Ccdc134−/− were
noted. Thus, these data further suggest that Ccdc134
deletion causes embryonic lethality. In addition, in contrast to
Ccdc134+/+ and Ccdc134+/−
embryos whose red-brown livers invariably filled a large portion of
the abdomen, the livers of Ccdc134−/− embryos
were considerably smaller (Fig.
4B). The total cell number per fetal liver was
~2×106 in Ccdc134+/+ livers and
~5×105 in Ccdc134−/− livers,
suggesting that Ccdc134 deficiency affects the number of
fetal liver cells and may lead to a defect in hematopoiesis
(Fig. 4C).

Whole-mount histologic sections of
Ccdc134−/− embryos demonstrated liver
abnormalities in later stage embryos. The fetal livers were
markedly smaller and hypoplastic (Fig. 5A). Additionally, compared with
their littermates, severe hemorrhage and overall brain
disorganization was detected in the Ccdc134−/−
embryo brain ventricular space (Fig.
5B). Concentric hypertrophy of the cardiac ventricular wall was
also present in Ccdc134−/− embryos, suggesting
increased vascular resistance (Fig.
5C). Furthermore, vasculature malformation was also noted,
although blood cells present in Ccdc134−/−
embryos appeared morphologically normal (Fig. 5D). However, the lungs of
Ccdc134−/− embryos and littermate controls
presented normal branching of the bronchioalveolar tree, with
progressive thinning of the alveolocapillary membrane and
flattening of the terminal sac epithelium (Fig. 5E). Structural changes in the
placenta, leading to altered hemodynamics or surface area available
for nutrient exchange, have been demonstrated to result in
reductions in growth, heart defects and perinatal morbidity
(14) (Fig. 5F). Given this, whether loss of
Ccdc134 altered the morphology of the placenta was
investigated. The placenta of Ccdc134−/− embryos
was thinner and poorly developed, and demonstrated a prominence of
labyrinth trophoblasts, which were arranged into poorly formed
maternal vascular spaces; however, the thin-walled capillary bed of
fetal circulation was ill defined (Fig. 5F).

Lethality due to reduced cell
proliferation and vascular defects in Ccdc134−/−
embryos

To clarify the causes of the liver abnormality in
Ccdc134−/− embryos, immunohistochemical analysis
was performed in transverse sections from
Ccdc134−/− embryos at E13.5. As demonstrated in
Fig. 6, CCDC134 expression was
initially examined in vascular endothelial cells and some
differentiated erythroid cells of fetal livers; however,
Ccdc134−/− embryos were identified by
immunostaining for the absence of CCDC134 protein (Fig. 6A). As the embryonic liver is the
main site of hematopoiesis in the second-half of murine gestation,
it appeared that poor oxygenation secondary to anemia may
contribute to embryonic demise (15). Erythroid cells of fetal livers
were detected using the antibody against Ter119, which is a surface
marker for differentiated erythroid cells (16). As demonstrated in Fig. 6A, no obvious difference was
observed between the fetal liver of Ccdc134−/−
embryos and wild-type embryos.

To further evaluate the existence of the vascular
integrity defect, immunohistochemical analysis with specific
antibody against Vegfr-2 was conducted, which is a type V receptor
tyrosine kinase, predominantly known to be expressed in vascular
endothelial cells (17). The
representative example of Fig. 6A
illustrates a discontinuous Vegfr-2-positive endothelial cell
barrier in fetal livers in Ccdc134−/− embryos
compared with Ccdc134+/+ and
Ccdc134+/− embryos, which may contribute to fetal
liver abnormality. In order to further investigate the influence of
Ccdc134 deficiency on cell proliferation in fetal livers, a
proliferation study was performed at E13.5. Ki-67 immunostaining
detection of cycling cells revealed a significant decrease of
Ki-67+ nuclei in fetal livers of
Ccdc134−/− embryos compared with wild-type
embryos (Fig. 6A and C).
Observations of Ccdc134−/− embryos support that
the phenotype of an abnormal fetal liver described at E13.5 above
is due to a significant decrease in proliferation and impaired
blood vessels.

Gross morphology suggested severe hemorrhage in the
brain ventricular space of Ccdc134−/− embryos at
E13.5. Vascular integrity defect was further analyzed with
anti-Vegfr-2 antibody. As demonstrated in Fig. 6B, Vegfr-2 was expressed by both
cerebral tissue and vessels in the forebrain, midbrain and
hindbrain of embryos at E13.5; however, compared with the
Ccdc134+/+ and Ccdc134+/−
embryos, overall brain disorganization in the
Ccdc134−/− embryo was observed. Additionally, a
significant reduction of vascular density was observed in the
Ccdc134−/− embryos compared with their
littermates (Fig. 6D). Taken
together, these findings suggest that Ccdc134 may be
associated with angiogenesis.

Discussion

The present study reported the generation of
complete Ccdc134 null mice by deleting the four coding exons
of Ccdc134 using a Cre-loxP system, which abolishes the
expression of CCDC134 protein. The present study also demonstrated
the CCDC134 expression pattern during embryogenesis. Furthermore, a
large number of postnatal mice were examined and no homozygous
Ccdc134 KO mice were identified, suggesting that complete
loss of Ccdc134 resulted in embryonic lethality.

The main processes involved in mouse embryonic
development include regional specification, morphogenesis, cell
differentiation, cell growth and the overall control of timing
(18). We speculate that the main
cause of mortality in Ccdc134−/− embryos at E13.5
was anemia, as severe hemorrhage in the brain and neural tube was
identified. Notably, mortality at this age corresponds to a
developmental period during which defects of the heart or placenta
often lead to embryonic lethality (19). Additionally, the placentas of
Ccdc134−/− embryos were thinner and poorly
developed and showed a prominence of labyrinth trophoblasts. The
cell numbers in Ccdc134−/− fetal livers were
significantly reduced compared with the number in wild-type livers.
Also, fewer Vegfr-2-positive endothelial cells and reduced cell
proliferation were demonstrated in fetal livers of
Ccdc134−/− embryos compared to that of wild-type
embryos. It is also interesting to note that
Ccdc134−/− embryos demonstrated broad areas of
extravasated blood associated with discontinuous and defective
blood vessels. Angiogenesis is important in embryonic development,
in which endothelial progenitor cells (EPCs) serve critical roles
(20). Furthermore, angiogenesis
is driven by newly formed EPCs migrating from the sites of
hematopoietic stem cell (HSC) development (21). Bone marrow begins to function as a
source of HSCs just before birth, whereas in embryogenesis,
multi-lineage hematopoietic progenitors exist in the extraembryonic
yolk sac at E8.25, and in the placenta and embryonic
aorta-gonad-mesonephros region at E10 (22). From E12 to birth, the fetal liver
is the main site for definitive HSC formation (23). These findings implied that
Ccdc134 may have a role in hematopoiesis and angiogenesis
during embryonic development. Therefore, conditional deletion of
Ccdc134 in hematopoietic stem cells (Vav-Cre) and
endothelial cells (Tie2-Cre) may be utilized to further investigate
the function of CCDC134 in the differentiation of HSCs and
angiogenesis.

In conjunction with our previous results (4), CCDC134 was identified to be a novel
partner of hADA2a protein, which was a core component of the yeast
alteration/deficiency in activation (ADA)/GCN5 HAT complexes to
facilitate the acetylation of nucleosomal histones (24). Additionally, CCDC134 may act as a
critical regulator of hADA2a stability and activity, and
participate in the PCAF complex via hADA2a to affect its HAT
activity, which acetylates H3K14 and H4K8 (24). Deletion of GCN5 and PCAF resulted
in embryo lethality between E9.5 and E11.5, indicating that PCAF
and GCN5 served important roles in embryogenesis (25). Also, GCN5 and PCAF had redundant
functions in mouse embryonic fibroblasts (26). Additionally, HAT PCAF/lysine
acetyltransferase 2B was an important factor of the Hedgehog
signaling pathway that served an important role in embryonic
patterning and development of various tissues and organs, as well
as in maintaining and repairing mature tissues in adults (27). During embryogenesis, the early
mammalian embryo was characterized by large-scale chromatin
remodeling, including changes in histone variant incorporation,
global changes in DNA, and histone tail modification (28,29). Histone H3 acetylation in the
nucleosome core occurred with different temporal kinetics during
mouse pre-implantation development, and further affected embryo
development (30). In addition,
various studies have demonstrated that angiogenesis is precisely
regulated by soluble growth factors and receptor-mediated signals.
Vegfr-2 is a key regulator of angiogenesis, and its expression and
function are regulated by acetylation under dynamic control of the
acetyltransferase p300 (31,32). Furthermore, PCAF acts as a master
switch in the inflammatory processes required for effective
arteriogenesis (33). Therefore,
the cause of embryonic mortality in the present study may be due to
defective hematopoiesis and angiogenesis, which likely cause
mortality due to failure of acetylation modification of key
regulators during embryo development.

In conclusion, the present results support the
conclusion that disruption of Ccdc134 expression in mice
leads to embryonic lethality. In addition, the present preliminary
studies suggested that Ccdc134 may be related to
hematopoiesis and angiogenesis, and further studies will be
performed. Thus, the Ccdc134 null line provides a critical
tool for determining the physiological roles of Ccdc134.
Furthermore, Ccdc134fl/fl mice will be important
for the analysis of Ccdc134 function in specific cell types
and the extension into analysis of Ccdc134 loss of function
in adult animals.

Acknowledgments

We thank Mrs. Weiyan Xv (Peking University Human
Disease Genomics Center, Beijing, China) for technical support. The
present study was supported by the National Natural Sciences
Foundation of China (grant no. 81372254), the National Basic
Research Program of China (grant no. 2013CB837201), Beijing Natural
Sciences Foundation (grant no. 7142082) and the National Science
and Technology Major Projects of New Drugs (grant no.
2012ZX09103301-032).